Project Summary/Abstract: CTDP1 Regulates FANCI and the Response to DNA Interstrand Crosslinks RNA polymerase II subunit A C-terminal domain phosphatase, CTDP1, is the only phosphatase in the human proteome that contains a BRCA1 C-Terminal (BRCT) domain. This modular domain only exists in a small and specialized group of proteins that mediate the DNA damage response (DDR), but the role of CTDP1 in this process has not been delineated. Analysis of the BRCT interactome has identified protein interactions that occur between CTDP1 and the Fanconi anemia (FA) protein FANCI, which is required for the resolution of DNA interstrand crosslink (ICL) lesions and resistance to ICL-inducing agents mitomycin c and cisplatin. Preliminary results indicate that CTDP1 expression promotes FANCI activation and represses cellular sensitivity to the ICL-inducing compounds mitomycin c and melphalan. Repression of CTDP1 expression is also associated with an increased accumulation of cells in S-phase of the cell cycle under both normal growth conditions and in response to ICL. CTDP1 expression is required to facilitate ICL-induced FANCI interactions with DNA repair proteins, including the DNA damage foci protein ?H2AX. These results represent the first reported protein interaction between FANCI and a phosphatase that can regulate its function. Thus, the central hypothesis of this research is that CTDP1 is required for the cellular response to ICL damage through the regulation of FANCI activation, and that targeted inhibition of CTDP1 can sensitize cells to ICL-causing chemotherapeutics. The Specific Aims of this project are: 1) To characterize the impact of CTDP1-mediated FANCI phosphorylations on FA pathway response to ICL; 2) To characterize the impact of CTDP1 inhibition on ICL sensitivity in an orthotopic model of breast cancer; and 3) To identify small molecule inhibitors of CTDP1 using high-throughput screening. These experiments will utilize phosphoproteomics to characterize the temporal regulation of phosphorylation sites on FANCI regulated by CTDP1 following DNA damage. Xenograft models of breast cancer will be used to determine the effects of CTDP1 inhibition on cancer growth, metastasis, and response to cisplatin using in vivo imaging. Virtual and physical high-throughput screening of potential CTDP1 BRCT-FANCI protein interaction disrupting compounds will be completed, and selected lead compounds will be validated western blot analysis of CTDP1 regulated phosphorylation events on FANCI and RPB1. Successful completion of this research will lead to a detailed understanding of the CTDP1-mediated regulation of FANCI and the ICL DNA damage response, which could be used to sensitize crosslinker resistant cancers to DNA damaging therapies.